Project Summary/Abstract Twelve pentamers of the HIV-1 CA protein have the critical function of shaping the fullerene cone geometry of the mature capsid, by occupying declinations or sharp points of curvature in the hexagonal capsid lattice, and thereby allowing the assembling protein shell to close. Apart from this structural role, no other function has been previously ascribed to the CA pentamer. Here, we propose a novel functional role for the HIV-1 CA pentamer. Our premise is that there are two distinct conformational states of the pentamer, one initially identified through in vitro assembly systems and the second observed as the average pentamer configuration in virions. Published studies and our preliminary data indicate that the two pentamer forms are distinguished by the configurations of two ligand binding sites: one for IP6, and another for phenylalanine-glycine (FG) containing host proteins such as NUP153 and other nucleoporins, CPSF6 and SEC24C. We propose that the distinct pentamer states define a molecular switch that modulates the post-entry structure of the capsid. We further propose that our pentamer switch hypothesis can explain poorly-understood capsid properties and functions, such as mechanisms that underlie capsid stability, uncoating and capsid inhibition. In this exploratory R21 project, our major goals are to define the structural and biochemical properties of the proposed pentamer switch and test the key prediction that conformational switching is coupled to ligand binding. In two Specific Aims, we will apply modern implementations of protein thermal profiling and cryoEM, on novel and established in vitro capsid model systems that range from soluble pentamers to assembled capsid-like particles. If proven, our novel and innovative pentamer switch hypothesis would significantly expand understanding of the molecular basis of HIV-1 capsid function and could inform the development of novel capsid-targeted antivirals.